Specific binding reactions, such as antibody-antigen interactions, have been used extensively in immunoassays to detect and/or quantify a variety of drugs or other compounds of interest present in biological fluids. Such immunoassays have been developed for the determination of polyvalent antigens, such as proteins, as well as haptens.
Haptens are by definition molecules too small to stimulate the production of antibodies when administered to an animal; that is, haptens by themselves are not immunogenic. However, it is well known in the art that haptens can be made imunogenic by covalently attaching (conjugating) them to an appropriate carrier molecule. Administration of such an immunogenic conjugate to an animal generally elicits the production of a spectrum of antibodies, some of which are directed against antigenic sites (epitopes) native to the carrier and some of which are directed against the attached hapten molecules. Appropriate carriers commonly contain poly(amino acid) segments and include polypeptides, proteins and glycoproteins. Specific examples of useful carriers are keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), .gamma.-globulins and plant globulins (for example, pumpkin seed globulin).
A variety of chemistries have been used for preparation of the immunogenic hapten-carrier conjugates necessary for raising anti-hapten antibodies. Since the native carriers generally contain a substantial number of lysine residues, each of which contains a reactive amine group, one particularly useful process utilizes the reaction of a hapten or hapten derivative containing amine-reactive functionality with the carrier. An important aspect of this approach is that hapten or hapten derivatives functionalized with a carboxyl group are readily converted to an appropriate amine-reactive intermediate.
Quinidine, the hapten of interest here, is a significant pharmacological compound. Its value as an antiarrhythmic drug was discovered through the chance observation that malaria patients with atrial fibrillation were occasionally "cured" of their arrhythmia while under treatment with these drugs. ##STR1##
In the absence of liver and/or kidney disease, the biologic half-life of quinidine is about 6 hours. Part of the drug is excreted unchanged by the kidney and the remainder is metabolized by the liver to products that do not have antiarrhythmic activity. Quinidine affects the rate and rhythm of the heart through direct effects on the heart muscle and through indirect effects on the conduction system. Quinidine reduces the excitability, conduction velocity and contractility of the myocardium.
Quinidine frequently produces unwanted gastro-intestinal side effects, which are somewhat dose-related. Monitoring the plasma drug level provides information helpful in maintaining adequate therapeutic levels while minimizing undesirable side effects.
However, native quinidine does not contain the amine-reactive functionality described above, so that it is necessary to synthesize an appropriate quinidine derivative. The C-10-C-11 double bond present in quinidine offers a particularly attractive place to start.
Kobayashi et al. (Journal of Polymer Science: Polymer Letters Edition, vol. 20, 85-90, Functional Polymers 9. Asymmetric Catalysis by New Cinchona Alkaloid Derivatives. Effect of C(3) Substituent On Asymmetric Induction, John Wiley and Sons, Inc., [1982]) disclose the synthesis of cinchona alkaloid derivatives having an organothio group at the C-11 position of quinidine. However, none of the derivatives prepared contained functionality appropriate to the objectives of the present invention.
International PCT application having International Publication No. WO 91/16322, published Oct. 31, 1991, and International PCT application having International Publication No. Wo 93/07142, published Apr. 15, 1993, disclose the use of quinidine and quinine derivatives as asymmetric hydroxylation catalysts. The compounds disclosed include a derivative of quinidine in which the C-11 position of quinidine is derivatized with a sulfonyl or sulfonyl linkage as a critical control agent. Similarly, these derivatives do not contain functionality appropriate to the objectives of the present invention.
Specifically, none of references cited mention or suggest the preparation or use of quinidine derivatives for synthesis immunogenic conjugates or reporter reagents (e.g., particle reagents) useful in immunoassays for quinidine.
There is a need for quinidine conjugates and their application to immunoassays for quantifying quinidine in a test sample and, especially, to quinidine derivatives useful for raising anti-quinidine antibodies to enable timely, precise monitoring of quinidine levels.